DIA-PASEF Proteomic Profiling of Aspergillus nidulans under MpkA-Dependent Iron Stress

Filamentous fungi play important roles in both biotechnology (as producers of valuable bioproducts) and in human health (as opportunistic pathogens). The fungus Aspergillus nidulans is a widely used model organism; however, proteome coverage remains below 15%, limiting comprehensive proteomic analyses. Here, we applied data-independent acquisition-parallel accumulation serial fragmentation (DIA-PASEF) for high-depth proteome profiling of A. nidulans. Using DIA-PASEF, we identified 3,904 proteins across biological triplicates grown in complex medium. This represents a > 140% increase in protein identifications with over 5-fold reduction in analysis time, expanding proteome coverage to 37%. We further integrated proteomic and phosphoproteomic analyses under iron-depleted conditions in an MpkA-deficient mutant (ΔmpkA). Deletion of mpkA resulted in extensive remodeling of metabolic and signaling networks, with ∼500 additional proteins and >1,800 phosphosites identified relative to the control. Differential expression and phosphorylation increased by more than an order of magnitude in the ΔmpkA mutant across Fe conditions. Gene Ontology analysis revealed expanded and distinct biological processes under iron limitation, reflecting adaptive responses to combined iron stress and MAPK disruption. Overall, this study establishes a high-coverage proteomic resource and demonstrates that iron limitation and MAPK pathway disruption enhance siderophore biosynthesis.